Preliminary field study of hepatic porphyrin profiles of Astyanax fasciatus (Teleostei, Characiformes) to define anthropogenic pollution

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Chemosphere xxx (2005) xxx–xxx

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Preliminary field study of hepatic porphyrin profilesof Astyanax fasciatus (Teleostei, Characiformes)

to define anthropogenic pollution

Leonidas Carrasco-Letelier a,*, Gabriela Eguren a,Franco Teixeira de Mello a, Phillip A. Groves b

a Faculty of Sciences, University of Republic, Piso 11, Igua 4225, Montevideo, Uruguayb Anadromous Fisheries Biologist, Idaho Power Company, P.O. Box 70, Boise, ID 83706, USA

Received 5 August 2004; received in revised form 10 June 2005; accepted 1 July 2005

Abstract

The implementation of eco-toxicological assessment in South America is presently limited due to significant scientificinformation gaps concerning native species and their potential use as biomarkers. Recently, a common southern hemi-sphere fish species, Astyanax fasciatus, has been pointed out as a potential bio-indicator to anthropogenic pollution.This is a small, abundant, Neotropical characid, which is widely distributed from Central America south, to the Riode la Plata Basin of western Uruguay. Our study found a statistically significant increase of coproporphyrin, uropor-phyrin and protoporphyrin concentrations in hepatic tissues of A. fasciatus collected from a stream segment with highanthropogenic disturbance (due mainly to agricultural derivatives and motor vehicle transportation activities).Although the area studied showed differences in up and downstream limno-chemical parameters, these differences werenot related to the increase of hepatic porphyrin concentrations. Based on the results of our study, we conclude thatA. fasciatus is a good bio-indicator of exposure to environmental contaminants, and we propose that this abundant fishspecies be considered as a sentinel organism for monitoring potential disturbances to freshwater ecosystems.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Astyanax; Porphyrins; Exposure biomarker; Pollution

1. Introduction

The use of fish as bio-indicators, or as biomarkers,can provide important information useful for describingchanges in water quality and environmental health of

0045-6535/$ - see front matter � 2005 Elsevier Ltd. All rights reservdoi:10.1016/j.chemosphere.2005.07.005

* Corresponding author. Tel.: +598 2 5258618; fax: +598 25258617.E-mail address: [email protected] (L. Carrasco-Lete-

lier).

watersheds (van der Oost et al., 2003). This eco-toxico-logical information can be the basis for bio-surveillanceprograms. Eco-toxicological information presentlyavailable in scientific literature comes from species (fish,amphibians, birds and mammals) that belong almostexclusively to northern hemisphere ecosystems; however,there is a paucity of knowledge concerning the biologyand ecological characteristics of southern hemisphere,neotropical species, especially fishes (Buti, 1995; Butiand Miquelarena, 1995; Vari and Malabarba, 1998;

ed.

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Lizama and Ambrosio, 2002). This implies a scientificinformation gap for southern hemisphere eco-toxicolog-ical parameters, and illustrates a need for research in thisarea to obtain relevant information required for bio-sur-veillance programs in South America.

To address this problem, several South Americancountries have initiated eco-toxicological studies to iden-tify species that might function as adequate bio-indica-tors (Schultz and Martins-Junior, 2000; Herkovitset al., 2002). Recent investigations have proposed theabundant and common characid, Astyanax fasciatus,as a potential bio-indicator species (Schultz and Mar-tins-Junior, 2000; Garcıa et al., 2002). This fish is widelydistributed from Central America south to the Rio de laPlata basin bordering northeastern Argentina and wes-tern Uruguay (Ringuelet et al., 1967). It is a pelagic fishthat inhabits streams and rivers without strong currents,as well as lentic systems, and which lacks migratorybehavior (Mackay, 1970; Vazzoler, 1992; Galvis et al.,1997; Riede, 2004).

In order to expand on the eco-toxicological parame-ters that might be assessed using A. fasciatus, we studiedthe feasibility of using hepatic porphyrin profiles (con-centrations) to document different degrees of environ-mental quality. Hepatic porphyrin concentrations area general biomarker of exposure that can identify spe-cific chemical groups belonging to environmental pollu-tants. Porphyrins are the intermediate metabolites ofheme biosynthesis. This metabolic pathway may be al-tered by environmental contaminants such as polychlo-robiphenyls (PCBs), dioxins and heavy metals leadingto changes in their concentration due to accumulationor excretion (Marks, 1985). For example, polyhalogen-ated aromatic compounds (such as PCBs and dioxins)lead to the accumulation of uroporphyrin (Mirandaet al., 1992; van Birgelen et al., 1996); herbicide pollu-tion promotes the accumulation of protoporphyrin(Bleakley et al., 1979; Leonzio et al., 1995); heptachlor,lindane, arsenic, and mercury result in the accumulationof coproporphyrin (Taira and San Martın de Viale,1980; Martinez et al., 1983; Woods and Southern,1989; Woods et al., 1991; Bowers et al., 1992; Nget al., 2002); while lead promotes accumulation of pro-toporphyrin and coproporphyrin (Hichiba and Tomo-kuni, 1987; O�Halloran and Duggan, 1988; Pain, 1989;De Matteis and Lim, 1994). These various authors val-idated porphyrins as indicators of exposure to contam-inants, confirming a close link between the contaminantand induction of specific porphyrins in target organs/tissues.

Freshwater monitoring programs presently in use inUruguay only analyze the physico-chemical status ofwater. This strategy is expensive, restricts its applicationto few water bodies within the country, and does notallow for the eco-toxicological qualification of theecosystem assessed. The continuing degradation of the

quality of Uruguayan freshwater systems, due to theirrational use of the national hydrographic net, pointsout the necessity to develop new tools for bio-surveil-lance programs (Praderi and Vivo, 1969; Arocena andPintos, 1988; Arocena et al., 1989; Arocena, 1996).Specifically, we believe it is necessary to design a moni-toring system of persistent organic pollutants (POPs)(Barra et al., 2002). In this respect, the identification ofbiomarkers within a common fish species can lead tothe implementation of a strategy that may provideresearchers and resource managers with the ability toidentify specific types of pollutants present, and to assesstheir biological effects within aquatic ecosystems.Considering the rapid reduction in overall water qualitywithin Uruguay, we believe that there is an urgent needto identify exposure biomarkers in order to address themonitoring of chemical pollutants. The purpose of ourwork is to define the capacity of hepatic porphyrinprofiles from A. fasciatus to assess environmental healthchanges due to non-point pollution sources.

2. Materials and methods

2.1. Study area

The general study area is located within the SantaLucıa River Basin (1435 ha) of southern Uruguay,which includes the potable water source of Montevideo(Capital City of Uruguay with 50% of the national pop-ulation, approximately 1500000 inhabitants) (DNH,1999). Our specific study system is the Canada delDragon creek, a small tributary within the lower SantaLucia River Basin (Fig. 1). This tributary has alength of 13.7 km, a mean slope of 0.54%, a maximumelevation of 60 m, a drainage area of 14.67 km2, andcontains soil types dominated by Argisols and Mollisols(Altamirano et al., 1976). This tributary can be classifiedas a stream of third-order (Strahler, 1986).

Canada del Dragon creek can be divided into twosub-reaches of almost equal length, each with differentenvironmental characteristics and pollution loads(Fig. 1). Zone A, the upstream sub-reach, has been sub-jected to 50 years of intensive agro-chemical application(mainly organophosphates and carbamates) (Machadoet al., 1992), introduction of heavy metals and polycyclicaromatic hydrocarbons (PAH), as well as runoffcontaminated by fossil-fuel combustion products fromheavy traffic on nearby Montevideo highways. Thevegetation cover of this zone is mainly comprised ofdeciduous fruit trees (SGM, 1988a,b). Zone B, the lowersub-reach, has a more natural vegetation covercomprised of prairies and wetlands, with small agricul-ture patches. This lower sub-reach has historicallyexperienced a reduced local load of agro-chemical andheavy metal pollution.

Fig. 1. Representative map of the study area. Highlighted areas (in black) indicate Uruguayan territory in South America (A);Montevideo region in Uruguay (B); Montevideo city and surrounding rural area (C); and Canada del Dragon creek basin (D).

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2.2. Chemical analysis

Water parameters such as pH, dissolved oxygen(mg l�1), conductivity (lS cm�1) and temperature (�C)were measured using portable meters. Alkalinity(mg CaCO3 l

�1) and particulate organic matter (mg l�1)were determined in the laboratory from water samplescollected in one litre plastic bottles (APHA, 1995). Con-currently, potential agro-chemical contaminants wereidentified in each zone (dose and application frequen-cies) through interviews with local landowners. Basedon their information, the approximate loading of agro-chemical contaminants (herbicide, insecticide, fungicide)used in the basin was estimated.

2.3. Biological sampling

Fish samples were collected during the spring of 2002(November within the southern hemisphere). Specimenswere captured with electric fishing equipment (TypeFEG 1000, Sachs Elektrofischfanggerate GmbH, Ger-many). Captured individuals of A. fasciatus were pre-served on ice until processing in the laboratory. Welimited our analyses to adult specimens (body weight

range 5–8 g; length 56–69 mm) obtained from each Zone(seven fish from zone A, and ten fish from zone B)(Fig. 2).

2.4. Biomarker analysis

The analysis of porphyrin concentrations can beaccomplished through non-destructive techniques(blood samples; Casini et al., 2001). However, in ourstudy this was not possible because the diminutive sizeof A. fasciatus (maximum length: 11 cm) (Ringueletet al., 1967) does not easily allow researchers to obtaina sufficient quantity of blood. For our study, the fishwere kept alive until sacrificed in the laboratory. The liv-ers of each specimen were immediately removed,weighed (to the nearest 0.0001 g), and individuallyhomogenized within distilled water at a ratio of 1 g tis-sue to 10 ml water. Equal samples (0.2 ml) of thehomogenates were then transferred to individual glasstubes containing 1.6 ml of a methanol/1 N perchloricacid mixture (50:50, v/v). After vortex-mixing, each sam-ple was kept in the dark for 10 min and then centrifugedfor 3 min at 15000 rpm. For quantitative determinationof porphyrins we used a fluorimetric assay described by

Fig. 2. Photograph of a typical adult Astyanax fasciatus (approximately 7 cm total length) captured in Canada del Dragon creek.

Table 1Active compounds of agro-chemicals used in Canada delDragon creek basin (information obtained from landownerinterviews)

Active compounds CAS number Use

Azinfos methyl 86-50-0 InsecticideChlorpyriphos 2921-88-2 InsecticideCypermethrin 67375-30-8 InsecticideDiazinon 333-41-5 InsecticideDimethoate 60-51-5 InsecticideDiphenilamine 122-39-4 InsecticideDNOC 534-52-1 Insecticide/

fungicideDodine 2439-10-3 FungicideEndosulfan 115-29-7 Insecticide

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Grandchamp et al. (1980). The final concentrations ofporphyrins were expressed in nmols/g tissue (wetweight).

2.5. Statistical analysis

We used Statistica 4.0 statistical software to performall statistical analyses. According to the Kolmogorov–Smirnov goodness-of-fit test all of the data conformedto normal distributions. With the data that satisfied sta-tistical parametric assumptions, we used a Student�s t-test for equal and unequal sample sizes. For data thatdid not fit parametric assumptions we used a Kolmogo-rov–Smirnov two-sample test (Zar, 1999).

Glyphosate 1071-83-6 HerbicideImidacloprid 105827-78-9 InsecticideMancozeb 8018-01-7 FungicideMCPA 94-74-6 HerbicideMethidathion 950-37-8 InsecticideMetiram 9006-42-2 FungicideParathion ethyl 56-38-2 InsecticidePhosmet 732-11-6 InsecticideSimazine 122-34-9 HerbicideZiram 137-30-4 Fungicide

3. Results and discussion

3.1. Agro-chemical information

Through interviews with local landowners we estab-lished that approximately 700 ha of deciduous fruit treecrops were under cultivation within the study area,mainly situated in zone A. Fruit tree cultivation corre-sponded to approximately 50% apple crops, 20% pears,20% peaches, and 10% other fruit crops. Moreover, thelandowner interviews indicated an approximate annualuse of 8960 kg of insecticides, 22575 kg of fungicides,and 2478 l of herbicides within the study area. The activecompounds found within those agricultural chemicalsare identified in Table 1. Our data agrees with previousinformation concerning potential agricultural pollutantswithin the study area reported by Machado et al. (1992)and SGM (1988a,b).

3.2. Limno-chemical results

Water samples from within the study area showedstatistically significant differences between zones A and

B in alkalinity, pH, conductivity, temperature, and dis-solved oxygen (Table 2). However, particulate organicmatter (POM), mean water velocity, mean stream depth,and stream width in each zone were not statistically dif-ferent (Table 2). The differences observed for alkalinity,pH, conductivity, temperature and oxygen dissolvedshowed the expected natural differences between up-stream and downstream sub-reaches of a lotic system(Vannote et al., 1980). However, these differences didindicate that our arbitrary division defined for our sam-pling design, based on landscape characteristics andhuman land-use, was reflected in the limno-chemicalparameters measured.

Table 2Mean values of limno-chemical parameters (±SE) in Zones A and B of Canada del Dragon creek

Limno-chemical parameters Zone A Zone B P-values of differencesMean ± SE (n) Mean ± SE (n)

Alkalinity (mg CaCO3 l�1) 136.50 ± 20.49 (6) 212.50 ± 37.28 (6) 0.001

pH 7.53 ± 0.06 (20) 7.88 ± 0.05 (20) 0.000Conductivity (lS) 768.85 ± 81.90 (20) 644.85 ± 36.64 (20) 0.001Temperature (�C) 20.20 ± 0.46 (20) 20.99 ± 0.31 (20) 0.004Dissolved oxygen (mg l�1) 4.36 ± 0.28 (20) 5.15 ± 0.13 (20) 0.000POM (mg l�1) 19.65 ± 9.37 (6) 18.08 ± 11.54 (6) NSWater velocity (m s�1) 0.23 ± 0.17 (10) 0.55 ± 0.60 (10) NSChannel depth (m) 0.29 ± 0.14 (10) 0.30 ± 0.11 (10) NSChannel width (m) 2.28 ± 0.95 (10) 3.06 ± 0.81 (10) NS

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3.3. Biomarker results

No statistical differences were found between theweights of liver tissue of A. fasciatus collected in zonesA and B. The comparison of hepatic porphyrin concen-trations of specimens from the two study zones showed ahighly significant difference in all porphyrins measured(Table 3). The Students� t-test for unequal sample sizesconfirmed that the mean concentration of protoporphy-rin within hepatic tissue of specimens from zone A was158.5 nmols/g greater (P = 0.010) than what was mea-sured from specimens of zone B (Table 3). It was alsodetermined that the mean concentration of uroporphy-rin within hepatic tissue was 61.1 nmols/g greater(P = 0.018) in specimens from zone A (Table 3). Finally,our results indicated that the mean concentration ofcoproporphyrin within hepatic tissue of specimens fromzone A was 50.7 nmols/g greater (P = 0.034) than thatobserved in specimens from zone B (Table 3).

The lack of published data about hepatic porphyrinlevels in Neotropical fishes makes it difficult to discussthe results obtained in absolute terms. However, theseresults can be used in relative terms to define a potentialchange in environmental health conditions, confirmingthe assumption that zone A experiences a greater pollu-tant load than does zone B. This assumption is furthercorroborated by the agro-chemical data provided fromthe landowner interviews. As previously explained, theincrease of each type of hepatic porphyrin correspondsto specific biochemical responses widely distributed in

Table 3Mean concentration of hepatic characteristics of Astyanax fasciatus s

Hepatic porphyrinsconcentrations

Zone AMean ± SE (n)

Liver mass 53.63 ± 23.20 (7)Coproporphyrin 239.18 ± 49.93 (7)Uroporphyrin 257.33 ± 57.32 (7)Protoporphyrin 634.72 ± 122.64 (7)

Porphyrin concentrations (±SE) are expressed in nmols per gram of

nature, and which can be linked to characteristic chem-ical groups (Marks, 1985; De Matteis and Lim, 1994).This biochemical knowledge allows researchers to inferwhich pollutant agents may affect the health of individ-ual species and to understand how local environmentalhealth conditions may be altered. Therefore, theincreased hepatic concentrations of protoporphyrin,uroporphyrin and coproporphyrin within specimens ofA. fasciatus from zone A indicate a condition ofincreased pollution in zone A from the local applicationof agro-chemicals, and from the incorporation of poly-halogenated aromatic compounds and heavy metals,respectively, into the local hydrosystem (Bleakleyet al., 1979; Hichiba and Tomokuni, 1987; O�Halloranand Duggan, 1988; Pain, 1989). Increases in pollutiondue to heavy metals is likely attributed to heavy trafficof highways present in zone A, while the polyhalogen-ated aromatic compounds and agro-chemical pollutionindicated by the biomarker agrees with the situation ofpesticide use in fruit crops mainly in zone A, andpossibly to other agro-chemicals listed in Table 1.

3.4. Integrative analysis

The biomarker results showed clear, statistically sig-nificant differences, indicating an increased pollutantload in zone A. However, because of the paucity of sci-entific information concerning the biology and ecologyof A. fasciatus, there remains the possibility thatthe changes detected could result from different, but

ampled in zones A and B of Canada del Dragon creek

Zone B P-values of differencesMean ± SE (n)

94.79 ± 38.62 (10) NS188.53 ± 29.87 (10) 0.034196.22 ± 28.91 (10) 0.018476.27 ± 74.72 (10) 0.010

hepatic tissue, and liver mass in milligrams.

Fig. 3. Principal component analysis results from correlation matrix of relevant parameters.

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natural, limno-chemical conditions. With respect to thispossibility, we assessed the relationship between thelimno-chemical parameters and biomarkers. To test thisassumption, we performed a principal component anal-ysis (PCA) based on a correlation matrix produced fromcomparison of all the parameters measured. The PCAresults indicated that the first two principal componentsexplained 71.7% of the total variance, and that therewere no relationships between porphyrins and limno-chemical parameters (Fig. 3). These results further agreewith scientific publications concerning porphyrin pro-files used as biomarkers (Fossi et al., 1995; Gavilanet al., 2001).

4. Conclusions

Our results clearly show that hepatic porphyrin pro-files obtained from specimens of A. fasciatus can per-form well as an exposure biomarker for the assessmentof several types of environmental pollution. Addition-ally, the use of this potential exposure biomarker isnot compromised by natural limno-chemical conditions.Our results illustrate the capacity of A. fasciatus for itsuse as a sentinel organism in freshwater monitoring pro-grams throughout its range in the southern hemisphere,and in particular within freshwater systems of Uruguay.Although the biomarker assessment made in this studywas accomplished using a destructive technique, theabundance and widespread distribution of A. fasciatusshould allow researchers, and the general public, to ac-cept the minor environmental impact that may result

from the sampling of this fish species for bio-surveillancemonitoring programs.

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